Polymerases are enzymes of fundamental importance to living organisms. They are responsible for the synthesis of nucleic acids and their transformation into other nucleic acids necessary for the synthesis of proteins. Polymerases are, therefore, found in all types of cells including the causative DNA virus for hepatitis B virus (HBV).
HBV is a cause of hepatitis, one of the most common infectious human diseases, and the resulting hepatocellular carcinoma (HCC) is one of the most common cancers worldwide. The most effective way in preventing HBV infection and disease to date is through immunization against HBV. The currently licensed HBV vaccines consist of the major surface antigen (HBsAg) in either its natural form (plasma-derived) or recombinant form (purified from yeast cells). The vaccine-induced antibodies have been shown to bind to the most antigenic xe2x80x98axe2x80x99 epitope located within residues 124 to 147 of HBsAg, resulting in effective neutralization of HBV replication.
Although such active vaccination programme has resulted in a decrease of HBV infection in the population, an increasing number of mutations located within the xe2x80x98axe2x80x99 epitope have been emerging. These vaccine-induced HBV mutants are of concern, as they are capable of escaping the currently available immuno-based diagnostic system and able to replicate independently.
The fact that mutations on the xe2x80x98axe2x80x99 epitope of HBsAg give rise to amino acid substitutions in the overlapping HBV DNA polymerase, particularly by their location within the reverse transcriptase domain, may imply that these vaccine-induced mutants have altered reverse transcriptase activity, a key factor for the viral replication.
HBVs are DNA viruses that replicate their genomes by reverse transcription of an RNA intermediate. Packaging of this RNA pregenome into nucleocapsides and initiation of the replication depends primarily on the interaction of the HBV DNA polymerase with the encapsidation signal. There are two copies of the encapsidation signals on the pregenomic RNA. Only the 5xe2x80x2 copy functions as a template for the priming reaction. Following initiation of the minus-strand DNA synthesis, the DNA oligomer (4 nucleotides) is transferred by an unknown mechanism to the 3xe2x80x2 end of pregenomic RNA, where it hybridizes to its complementary sequences. Reverse transcription then continues toward the 5xe2x80x2 end of the RNA template. The interaction between the HBV DNA polymerase and the pregenomic RNA occurs as a covalent link between the N-terminally located tyrosine residue on the DNA polymerase and a specific nucleotide within the encapsidation signal. The HBV reverse transcriptase is one of the four domains within the large DNA polymerase. Other domains include: i) the N-terminal protein, which is responsible for the covalent association of the polymerase to the pregenomic RNA; ii) the spacer region which is tolerant for mutations; and iii) the RNAse H domain involved in the degradation of the in RNA intermediate.
Similarly to other Polymerases, detection of the HBV DNA polymerase activity in vitro can be used in the following three situations:
Characterization of a newly isolated virus as a replicative virus and assess the differences with regard to other known viruses. This is particularly relevant for HBV variants with mutations on their surface antigens;
Determination of isolation success of virus from the test material of a subject known to be infected;
Assessment of in vitro efficiency of inhibitors of polymerases that may be antiviral agents.
Various systems have been established to measure the HBV DNA polymerase activity in vitro in the absence of viral replication and other viral proteins. One interesting finding has been the fact that a detectable priming activity of HBV DNA polymerase requires not only the N-terminal protein but also a functional reverse transcriptase domain. One common feature of these systems is therefore the detection of the priming activity of HBV DNA polymerase, indicative of HBV DNA polymerase activity. Two of such systems utilize the duck HBV (DHBV) DNA polymerase, and both have demonstrated reverse transcriptase activity that is template dependent and protein primed. One of the DHBV systems utilizes in vitro translation of DHBV DNA polymerase to obtain lysates that contain a functional DNA polymerase, while the other system packages an active fusion protein of DHBV DNA polymerase in a virus-like particle from the yeast retrotransposon Tyl. Active DNA polymerase has been measured by its priming activity, as indicated by the radio-labeled protein in the presence of priming nucleotide (i.e. [xcex1-32P] dGTP for DHBV DNA polymerase). A similar activity assay has recently been reported for the human HBV DNA polymerase. A 350 base pairs 3xe2x80x2 non-coding region of the polymerase containing the encapsidation signal has been included in all reported constructs, therefore pointing to its importance for in vitro activity assay.
One direct application of the in vitro activity assay for human HBV DNA polymerase may be the screening of novel antiviral agents. Antiviral therapy of chronic HBV infection still remains a problem since several clinical trials have shown that a sustained response to interferon or nucleoside analogs is observed in only 30 to 40% of the patients studied. This response rate is even lower in long-term HBV carriers and in immunocompromised patients. The design of new protocols for chemotherapy to eliminate HBV is needed since the majority of the patients will not clear the viral infection and, therefore, will be at great risk of developing progressive liver disease and hepatocellular carcinoma (HCC). It will also be of particular interest to assess the antiviral effects of such agents on human HBV surface antigen mutants.
Limiting factors exist, however, for antiviral testing using the established in vitro activity assays for human HBV DNA polymerase. One of these has been the cloning step that places the coding region of the HBV DNA polymerase under the control of a viral polymerase promoter (e.g. SP6 or T7) on a plasmid (i.e. pGEM-T) is required in all established systems. In addition, HBV DNA polymerase expressed in some systems requires further purification (i.e. immunoprecipitation) prior to its activity assay. These tedious manipulations are not practical in view of the large number of HBV mutants.
According to the first aspect of the present invention, there is provided an in vitro activity assay for human hepatitis B virus (HBV) DNA polymerase, which comprises using, as the 5xe2x80x2 oligonucleotide in PCR amplification of HBV DNA polymerase from a sample, an oligonucleotide into which has been incorporated the SP6 viral polymerase promoter, directly transcribing and translating the PCR products in eucaryotic cell-free lysates and measuring the priming of the HBV DNA polymerase in the presence of a radio-labelled agent.
According to a second aspect of the present invention, there is provided the use of an assay according to the first aspect of the invention to assay activity of various serum samples and/or to screen for inhibitors of the HBV DNA polymerase.
According to a third aspect of the present invention, there is provided the use of an assay according to the first aspect of the invention to test and/or screen potential anti-HBV drugs for their ability to inhibit DNA priming activity of human HBV DNA polymerase. Such a use preferably includes the steps of:
a) preparing at least one sample in a defined volume of assay buffer, each sample containing in vitro translated human HBV DNA polymerase protein in eukaryotic cell-free lysate using linear PCR product as template, an RNA template for HBV minus strand synthesis which includes a sequence capable of forming a stem loop structure,
b) preparing a control sample in an equivalent volume of the assay buffer, the control sample containing in vitro translated human HBV DNA polymerase protein using linear PCR product as template and an RNA template for HBV minus strand synthesis which includes a sequence capable of forming a stem loop structure, essential for binding and priming of the human HBV DNA polymerase,
c) incubating the samples to obtain a complex comprising DNA polymerase and the radio-labelled agent,
d) separating the complexes from the assay buffer, and
e) measuring the amount of radio-labelled agent in each separated complex, the amount being indicative of the DNA priming activity of the human HBV DNA polymerase.
Step c), d) and e) above may be replaced by:
c) incubating each said test tube and said control tube with equivalent amounts of a radio-labeled nucleotide triphosphate comprising the first nucleotide incorporated into said HBV minus strand DNA, under defined conditions favorable to DNA priming activity of said human HBV DNA polymerase, resulting in the formation of a complex comprising said DNA polymerase and said radio-labeled nucleotide triphosphate;
d) separating said complex from said assay buffer in each said test tube and said control tube by filtration through nitrocellulose membrane;
e) measuring the amount of said radio-labeled nucleotide triphosphate in each said separated complex, said amount being indicative of said DNA priming activity of said human HBV DNA polymerase;
f) comparing the respective amounts of said radio-labeled nucleotide triphosphate in said separated complexes which remained bound to the nitrocellulose membrane of each said test tube and said control tube, a decrease in the amount of said radio-labeled nucleotide triphosphate in said separated complexes of said test tube as compared to said control tube being indicative of inhibition of said DNA priming activity of said human HBV DNA polymerase by said potential anti-HBV drugs.
The present invention concerns a simplified activity assay for the hepatitis B virus (HBV) DNA polymerase, which assay is potentially more rapid than previous assays. The method involves the incorporation of the SP6 viral polymerase promoter into an oligonucleotide, which is then used as the 5xe2x80x2 oligonucleotide in PCR amplification of the HBV DNA polymerase from serum samples. The PCR products, containing the entire coding region and a 300 bp 3xe2x80x2 non-coding region with the encapsidation signal, are directly transcribed and translated in the wheat germ cell-free extract. The priming of the HBV DNA polymerase to the intermediate RNA template, the key indicator of its enzymatic activity, is measured in the presence of the radio-labeled [xcex1-32P] dTTP. Applications of the present invention cover the activity assay from various serum samples (including those of HBV with mutations on the major surface antigen), and screening for inhibitors of the HBV DNA polymerase.
The invention provides a simplified process for assaying in vitro the human HBV DNA polymerase activity directly from the linear DNA template amplified from serum samples. The protein involved in the activity assay is generally produced in the wheat gem cell-free system by coupling the transcription and translation from the linear DNA template, which contains a SP6 viral polymerase promoter at its 5xe2x80x2 end.
For the achievement of this and other objects, the present invention provides a method for producing the HBV DNA polymerase directly from linear DNA fragment amplified from serum sample. In the presence of the radiolabeled agent, such as [xcex1-32P] dTTP, the resulting protein is assayed for its priming activity, a crucial step in hepatitis B viral replication.
The present invention provides a simple, sensitive and rapid assay that is specific for HBV DNA polymerase activity. The present invention includes a method detecting HBV DNA polymerase activity from serum samples comprising:
One-step extraction of HBV viral DNA from serum using phenol/chloroform;
PCR amplification of the coding region of HBV DNA polymerase (2400 base pairs) and its 3xe2x80x2 non-coding region (310 base pairs). The 5xe2x80x2 oligonucleotide includes a 5xe2x80x2 proximal SP6 viral polymerase promoter;
In vitro translation of HBV DNA polymerase with PCR amplification products as template;
Activity assay on extracts containing the translated HBV DNA polymerase and in the presence of radiolabeled priming nucleotide.
Further subject matters of the present invention include reagents for implementing the method, a method of determining the inhibitory effect of substances on polymerase activity and an application on polymerase activity of HBV variants with mutations on the surface antigen.
In accordance with the present invention, serum sample may contain a HBV variant with unspecified DNA polymerase activity. The present invention enables its qualitative determination. For cases whereby quantitative measurements of the DNA polymerase activity are required, the activity assay in the present invention can be further developed (i.e. measurement of the primed DNA polymerase on nitrocellulose membranes). For cases whereby inhibitory effects of a molecule on the DNA polymerase are to be determined, the amount of primed DNA polymerase can be compared to that produced from a control sample where no inhibitory molecule is present.